Numerous bacterial, viral, and parasitic infections of mammals have two phases of infection: an acute phase during the early stages of the infection, sometimes followed by a prolonged chronic phase having a finite or indefinite duration. The ability of an infectious agent to establish a chronic infection in a mammalian host depends to a significant extent on the capacity of the host immune response to eliminate the infecting organism from the host in the early stages of the infection. The specific immune mechanisms responsible for eliminating the infectious agent from the host differ depending on the infectious agent. In the case of viral and some parasitic infections, the infectious agent-eliminating activity of cytotoxic T lymphocytes is believed to comprise a pivotal component of the host immune response for mediating the elimination of these agents from the host.
The components of a mammalian immune system to which mammalian immune response activities can be attributed include, but are not limited to, antibody molecules, complement molecules, B lymphocytes, T lymphocytes, cytotoxic T lymphocytes, helper T cells, suppressor T cells, immunosuppressive lymphocytes, cytokine-secreting lymphocytes, other non-cytotoxic lymphocytes, macrophages, neutrophils, mast cells, basophils, eosinophils, monocytes, and the like. Induction or replication of the host immune activities leading to complete elimination of an infectious agent from a mammalian host is the paradigm for a clinical treatment for infection by a infectious agent.
In the course of infections with bacteria and some parasites, elimination from a host of an infectious agent causing an acute infection has traditionally been accomplished using antibiotics which serve as relatively selective poisons for the infectious agent. Antibiotic treatment has been less successful in the case of chronic bacterial infection. More recently, clinical efforts have focused on modulating the host immune system in an attempt to eliminate infectious agents causing chronic infections in cases wherein indolence of the host immune system contributes to persistence of the infectious agent. Specific immune modulation using substances such as interferons alpha, beta, and gamma has been attempted, and in a minority of cases beneficial results have been observed.
When an infection becomes chronic, the infection may be controlled by a persistent host immune reaction to the infectious agent. Certain herpes viruses, for example, remain latent only in the context of host immune competence. Immunosuppressive therapy used, for example, in organ transplant recipients permits latent herpes virus to become reactivated. Thus, loss of immune competence in response to steroid and cyclosporin A administration to a human patient having a latent HHV-6 infection permits recrudescence of HHV-6. The result of HHV-6 reactivation includes viral pneumonia and bone marrow suppression. In addition, the high incidence of non-Hodgkins B cell lymphomas among humans infected with the AIDS virus (HIV-1) demonstrates that pathogenicity attributable to chronic Epstein-Barr virus infections becomes active as T cell competence is lost. Thus, reactivation of pathogenicity attributable to an otherwise non-pathogenic chronic infection which is effected by suppression of the host's immune competence may have deleterious effects on the host.
The mucosal immune system includes those immune cells and organs directly associated with the mucosal lining of the gastrointestinal tract and lungs, including the airways. Such a partitioning of the body's total immune network is not arbitrary, but is based on certain widely recognized unique features of the mucosal immune system. One of the distinguishing features separating mucosal immunity from other types of immunity is the requirement that the mucosa physically transports, or permits ingress of foreign substances into the body. In the gut, nutritional substances must be absorbed for metabolic purposes. Many, and probably most of the absorbed nutrients are antigenic. Proteins, lipoproteins, carbohydrates complexed with proteins, and the like can and do stimulate immune priming and subsequent recognition. Further, because of the large concentration of symbiotic (but potentially pathogenic) microorganisms in the gastrointestinal tract, the mucosal immune system must prevent adverse immune reactions that would result in elimination of the symbionts through inflammatory immune recognition.
Another distinctive feature of the mucosal immune system is the ability to generate a specific isotype of antibody (IgA) which can prevent absorption of antigen but is non-inflammatory. While certain sub-classes of IgG are similarly non-inflammatory, IgA is predominantly localized to the mucosal immune system.
It is also recognized by those skilled in the art, that the mucosal immune system is the numerically dominant member of somatic immunity. Studies have shown that each meter of human intestine contains approximately 1012 lymphocytes. On the basis of immunoglobulin-secreting cells, the human gut contains several-fold more cells than the remainder of the immune system. Additionally, the phenotypic character of the immunocytes populating the mucosal immune system is distinctive. Murine and human intestine contain a large percentage (up to 90%) of an atypical lymphocyte; the gamma-delta T lymphocyte. These cells are unique because they are not educated in the thymus during ontogeny as are the majority of alpha-beta T cells populating the spleen, peripheral lymph nodes, or circulation.
Yet another feature of the mucosal immune system that separates it from the somatic immunoaxis is the presence of predominantly anti-inflammatory T helper 2 cytokines. The tissue supporting the single-cell barrier separating the lumen of the gastric or respiratory system from the external milieu, the lamina propria, is rich in interleukin-4, interleukin-10, and transforming growth factor-b. It is known to those skilled in the art that immune cells antigenically stimulated in such an environment differentiate into immune effectors with anti-inflammatory (TH2) functions. However, such an environment does not guarantee ultimate TH2 function, and this has been shown to occur in the mucosal immune system. Specifically, antigen presentation to the mucosal immune system typically has three outcomes as set forth below.
1. When an antigen is transported across the mucosal epithelium, it can be processed by immune accessory cells, which may migrate to local immune accessory organs (regional lymph nodes) and there cognitively present antigen or antigen fragments to other immune cells resulting in the numerical expansion of antigen-reactive lymphocytes. A proportion of the activated lymphocytes mature to plasma cells that manufacture the mucosal-dominant, non-inflammatory immunoglobulin IgA. Another proportion of the cells differentiates into inflammatory (TH1) cells such as IgG2a secreting plasma cells, interferon-gamma or Interleukin-2 expressing T helper lymphocytes.
2. Some of the processed antigen is presented to immature B lymphocytes that differentiate into IgA secreting plasma cells. These cells migrate back to the mucosa where they establish residence, and express the specific immunoglobulin. The majority of the immune protein is then transported into the lumen of the gut or lungs where it reacts with epitopes of the educating antigen. Resultant antibody binding to the immunogen can prevent transmucosal ingress.
3. The mucosal immune system, perhaps due to its unique physiological responsibilities, concomitantly drives one of two additional immunological events. These events are not mutually exclusive, and are believed to occur in a continuum dictated by the amount of antigen administered as well as dose timing, frequency, and simultaneously administered proteins. The first of these events is the education and expansion of another arm of the immune system (low dose tolerance).
When small (ca. unit milligram) amounts of the antigen are presented to the mucosa, a set of lymphocytes is enabled for education and expansion. The distinguishing characteristic of this subset of educated cells is their ability to accept a biochemical signal of active inflammation and recognize the presence of the original educating antigen in intimate physical proximity to sites of inflammation. When both activation criteria are present, the suppressor lymphocytes express certain immune regulatory cytokines resulting in an active down regulation of the inflammatory reaction. These biochemicals include interleukin-4, interleukin-10 and TGF-B.
A second mechanism of tolerance engendered by antigen presentation to the mucosal immune system is referred to as “high-dose tolerance”. This mechanism comes into play following administration of larger (ca. 10 mg or larger) amounts of the educating antigen. “High dose” tolerance appears to directly disable the antigen-reactive lymphocyte. It has been suggested that the administration of large amounts of antigen results in a reduced proteolytic processing of the antigen in the gut or lungs with an increased concomitant availability of non-degraded antigen which is transferred across the mucosal barrier intact. Reactive lymphocytes bind large amounts of the antigen, and are rendered anergic perhaps because the stoichiometry of antigen presentation with required activation signal, although other explanations have been offered. Experimentally, the two mechanisms are differentiated by the demonstrable requirement for educating antigen and presence of IL-4, IL-10, and TGF-B in immunosuppressed cultures or animal models in the first instance. In contrast, anergy is experimentally demonstrated by reversal of immune unresponsiveness by the addition of exogenous IL-2, and the relative paucity of suppressive cytokines present in active suppression.
Published work from several laboratories has confirmed that both of the above-described mechanisms may operate concomitantly. The foregoing discussion is provided to clarify the known mechanisms of oral tolerance, and not to differentiate them. Both operate to suppress immune responsiveness to mucosally presented antigen, and appear to be functional in the suppression of inflammatory responses to inhaled or eaten foreign substances.
Virtually all mucosally presented antigens elicit tolerance. Low dose tolerance occurs following the administration of myelin basic protein in a model of multiple sclerosis, type II collagen in a model of rheumatoid arthritis, retinal S antigen in a model of autoimmune uvitis and insulin in a model of type I diabetes. Further, mice fed recombinant acetylcholine end plate receptor protein have been found to be refractory to immune stimulation with the same protein in a model of myasthenia gravis. Additionally, oral ovalbumin administration has been shown to elicit tolerance in ovalbumin-TCR transgenic mice. High dose tolerance has also been reported for some of the same as well as different proteins.
There are certain reported exceptions in the otherwise universal ability of orally or nasally administered proteins to establish oral tolerance. These exceptions may be divided into two categories based on either the biochemical properties of the administered protein or the physical form of the antigen. The oral administration of cholera holotoxin resulted in systemic immunity based on several experimental criteria. Similarly, the mucosal exposure of mice to the heat labile enterotoxin of certain E. coli strains resulted in the presence of circulating IgG antibodies reactive with the protein. Both of these substances belong to a unique class of toxins that are known to activate phosphorylation of specific transmembrane receptors present in enterocytes in the mucosa. Additionally, the diseases caused by the microorganisms that produce these proteins are enteric, and thus, the proteins are known to be naturally pathogenic at the mucosal level.
The second form of exception to orally administered proteins inducing an immune response instead of tolerance concerns the physical form of the presented antigen. Arntzen and colleagues reported the presence of systemic antibodies reactive with the nucleocapsid of Norwalk virus after feeding extracts of tobacco containing the transgenically expressed virus product (Arntzen et al., 1996, PNAS 93(11):5335-40). These researchers also discovered the presence of systemic immunity to hepatitis B surface protein (HBsAg) in mice fed repeated doses of following isolation of the protein expressed in a similar transgenic plant system (Kong et al., 2001, PNAS 98(20):11539-44.). However, the simultaneous administration of the mucosal adjuvant cholera holotoxin was required for maximal effect. A minor response was induced by feeding primed mice the plant extract alone, perhaps due to the physical form of the antigen. Importantly, apparently intact 17 nM pseudovirions were seen in homogenates of the plants, indicating that the transgenic protein self-assembled within the plant tissues
Another example of the ability of orally presented antigens to elicit an immune response is found in the recent work of Koprowski and colleagues. These investigators inserted the coding sequences for two protective epitopes from the rabies virus into alfalfa mosaic virus coat protein (CP), and then rescued the transgenic protein by enabling viral replication in plants in a complemented system using either tobacco mosaic virus lacking native CP(A4-g24), or in trans following infection with infectious alfalfa mosaic virus. Mice fed the chimeric viral particles were found to develop a systemic immune responsiveness (Yusibov et al., 2002, Vaccine 20(25-26):3155). Kapusta et. al. engineered a commercial cultivar of tomato to express the coat protein of the rabies virus, and reported that mice fed freeze-dried fruit became immune to challenge with normally lethal concentrations of the virus (Kapusta et al., 2001, Adv. Exp. Med. Biol. 495:299-303). Additional work by these researchers provided evidence that tomato's expressing the HBsAg could elicit circulating antibodies in mice formally adequate to provide immune protection from infection based on established IgG concentrations in humans (Kapusta et al., 1999, FASEB J. 13(13):1796-9). These results, collectively, suggest that oral administration of certain vaccine antigens results in the boosting of immunologically primed animals, or in rare cases the establishment of protective immunity in immunologically naive mice, but not the anticipated immune indolence of mucosal tolerance. An important common feature of these results is that the transgenically expressed antigen was either biochemically constrained to assemble into macromolecular structures, or was detected as complexed with plant organelles such as Golgi bodies, vesicles, plasma lemma and cell walls; structures effectively forming macromolecular complexes.
There is a long felt need in the art for efficient methods of immunizing a patient against an antigen. There is also a need for a method of presenting antigens to efficiently induce mucosal tolerance. The present invention meets these needs.